Protocol for the isolation of mouse senescence-associated CD4+ T cells using flow cytometry and functional assays

Summary Senescence-associated (SA) CD4+ T cells, which increase with age, may underlie the development of autoimmunity and chronic inflammation, but their pathological function remains understudied. Here, we present a protocol to isolate CD153+ SA-T cells and evaluate their characteristic responses upon T cell receptor stimulation. We describe steps for the isolation of CD153+ SA-T cells using flow cytometry and in vitro culture with stimulatory antibodies against CD3, CD28, and CD153. We then detail the assessment of the proliferation capacity and cytokine production. For complete details on the use and execution of this protocol, please refer to Fukushima et al. (2022).1


SUMMARY
Senescence-associated (SA) CD4 + T cells, which increase with age, may underlie the development of autoimmunity and chronic inflammation, but their pathological function remains understudied. Here, we present a protocol to isolate CD153 + SA-T cells and evaluate their characteristic responses upon T cell receptor stimulation. We describe steps for the isolation of CD153 + SA-T cells using flow cytometry and in vitro culture with stimulatory antibodies against CD3, CD28, and CD153. We then detail the assessment of the proliferation capacity and cytokine production. For complete details on the use and execution of this protocol, please refer to Fukushima et al. (2022). 1

BEFORE YOU BEGIN
Senescence-associated (SA) T cells were initially identified as programmed cell death protein 1 (PD-1 + ) CD44 hi CD4 + T cells. 2 Recently, however, SA-T cells expressing CD153 (CD153 + SA-T cells) have garnered increasing attention because of their potential relationship to the pathogenesis of systemic lupus erythematosus, type 2 diabetes, and chronic kidney disease. 1,[3][4][5][6][7] In response to T cell receptor (TCR) stimulation, CD153 + SA-T cells have diminished proliferation capacity and produce large amounts of a proinflammatory cytokine osteopontin (OPN), at the cost of typical T cell cytokines such as interleukin 2 (IL-2). Interestingly, concomitant engagement of CD153 enhances TCR signaling and upregulates the proliferation capacity and cytokine production of CD153 + SA-T cells.
This protocol describes the isolation and subsequent evaluation of CD153 + SA-T cells. Specifically, CD153 + SA-T cells were isolated from spleens using a flow cytometry-based method, and the TCRmediated response was assessed in vitro using plate-bound anti-CD3ε (a component of the TCR) and soluble anti-CD28 antibodies (Abs) (Figures 1A and 1B). The additional presence of plate-bound anti-CD153 Ab (clone RM153) served to recapitulate the enhancement of TCR signaling ( Figure 1B).
To facilitate the stimulation with plate-bound Abs, anti-CD3ε or anti-CD153 Ab is not used at the isolation step of CD153 + SA-T cells. We found that the majority of lymphocyte-activation gene 3 (Lag3 + ) CD121b -CD25 -PD-1 + CD44 hi CD4 + T cells were identical to the CD153 + SA-T cell fraction on a flow cytometry plot and thus were considered enriched CD153 + SA-T cells, 1 which enabled us to concentrate them without using anti-CD153 Ab. In this protocol, CD153 + SA-T cells are first isolated by either method A or B below.
A. Stain the splenic CD4 + T cells with staining panel A which lacks the anti-CD3ε Ab (Table 1), and isolate CD153 + SA-T cells with a straight gating strategy ( Figure 2A). B. Stain the splenic CD4 + T cells with staining panel B which lacks both anti-CD3ε and anti-CD153 Abs (Table 2), and isolate ''enriched'' CD153 + SA-T cells with an enrichment gating strategy ( Figure 2B).
Next, stimulate the isolated cells with stimulatory Abs in vitro. To examine their response to TCR stimulation (anti-CD3ε/CD28 Abs), the cells isolated by method A (or B) can be used, whereas to examine TCR stimulation with concomitant CD153 engagement (anti-CD3ε/CD28/CD153 Abs), method B should be chosen. Finally, the proliferation capacity and OPN production are assessed by fluorescence-activated cell sorting (FACS) and the enzyme-linked immunosorbent assay (ELISA), respectively. To confirm the characteristic responses of CD153 + SA-T cells, it is recommended to isolate and stimulate PD-1 -CD44 hi CD4 + T cells at the same time for comparison.

Institutional permissions
All animal experiments were approved by the Kyoto University Animal Research Committee and performed according to the Regulations on Animal Experimentation at Kyoto University. Permissions must be obtained from the relevant institutes prior to the animal experiments. . S1 and C1 indicate wells for CD153 + SA-T cells and PD-1 -CD44 hi cells, respectively. S2/3 and C2/3 indicate wells for biological replicates of S1 and C1, respectively. (B) Example of a 96-well plate layout for stimulation via CD3ε/CD28/CD153 (n = 3). S1 and C1 indicate wells for enriched CD153 + SA-T cells and PD-1 -CD44 hi (Lag3 -CD121b -CD25 -) cells, respectively. S2/3 and C2/3 indicate wells for biological replicates of S1 and C1, respectively.
(A and B) The diluted Abs or buffers and corresponding well positions are shown in the same color.  Figure 1A), dilute the anti-CD3ε Ab in phosphatebuffered saline (PBS) to a final concentration of 10 mg/mL. b. For stimulation by anti-CD3ε/CD28/CD153 Abs ( Figure 1B), dilute anti-CD3ε and anti-CD153 Abs in PBS to final concentrations of 10 and 25 mg/mL, respectively. Make a negative control of the CD153-mediated effects by diluting anti-CD3ε and rat IgG (instead of anti-CD153 Ab) in PBS to a final concentration of 10 and 25 mg/mL, respectively.
Note: Prepare the Abs in a sterile hood. Anti-CD28 Ab is unnecessary at this step. It is mixed with the cell suspension at a cell culture step.
2. Add 100 mL diluted Abs and PBS (non-stimulation control) to a flat-bottom 96-well plate ( Figures 1A and 1B). Add 200 mL PBS to the outer wells. As necessary, make biological replicates. PI is used to discriminate dead cells. Although almost all of the cells should be CD4 + , possible contamination of B220 + or CD11b + cells including minor CD4 + cells is removed. In a CD44 hi cell gate, CD153 + SA-T cells (CD153 + PD-1 + ) are frequently found. (B) Enrichment gating strategy employed without using anti-CD153 Ab. Lag3 + CD121b -CD25cells in a PD-1 + cell fraction concentrates CD153 + SA-T cells. 1 (C) Confirmation of successful sorting.
(A-C) A flow cytometer with 488/561/633/405 lasers was used. It should be noted that, unlike this laser system, both PI + dead cells and PerCP-Cy5.5labeled B220 + /CD11b + cells were removed together with PerCP-Cy5.5 when 488/633/405/355 lasers were used.   Make serial dilutions of the supernatant and determine an optimal dilution for blocking of the Fc receptors. Aliquot and store at 4 C for at least 3 months or À80 C for at least 3 years in a sterile condition.
Propidium iodide (PI) 100 mg/mL Dissolve 10 mg PI in 10 mL PBS and store in aliquots at À30 C (1 mg/mL stock). Furthermore, dilute the 1 mg/mL stock 1:10 in PBS and store in aliquots at À30 C (100 mg/mL stock). Frozen stocks are stable for at least 5 years. For use, thaw an aliquot of the 100 mg/mL stock and store at 4 C for at least 6 months protected from light.

Scissors and tweezers
Autoclave the scissors and tweezers and dry.

STEP-BY-STEP METHOD DETAILS
Purification of CD4 + T cells CD4 + T cells are purified from the spleens of aged mice with a magnetic cell separation system.
1. Sacrifice two mice by CO 2 asphyxiation and spray them entirely with alcohol disinfectant.
Note: If three biological replicates (n = 3) have been prepared, use a total of six mice; two mice for each pooled sample. Of course, the number of mice per pool should be changed according to the number of CD153 + SA-T cells needed. See also Expected Outcomes.
2. Collect the spleens. a. In a sterile hood, collect the spleens in a 70 mm cell strainer on a 6 cm petri dish filled with 5 mL FACS buffer. b. Mash the spleens with the plunger end of a sterile 1 mL syringe. Grasp the cell strainer using a tweezer and wash the suspension in the strainer with an additional 5 mL FACS buffer. c. Transfer the total 10 mL of suspension to a 15 mL conical tube and centrifuge at 350 3 g for 5 min at 4 C. d. Aspirate the supernatant.
CRITICAL: All steps from tissue collection to cell culture must be performed under sterile conditions. Alternatives: Instead of CD4 MicroBeads, a purification kit for unlabeled CD4 + T cells is also useful (e.g., mouse CD4 + T Cell Isolation Kit; Miltenyi #130-104-454).
6. Purify CD4 + T cells by magnetic cell separation. a. According to the manufacturer's instruction, place an LS column (Miltenyi) in the magnetic field of the MACS separator and rinse the column with 3 mL MACS buffer. b. Transfer the cell suspension to the column. c. Wash the column twice with 3 mL MACS buffer. d. Remove the column from the magnet and place on a 15 mL conical tube. Flush out the labeled CD4 + cells with 5 mL MACS buffer. e. Centrifuge at 350 3 g for 5 min at 4 C and aspirate the supernatant.
Optional: After flushing, count the cells using a hemocytometer. A rough estimation is that the total cell number will be 1.5-3 3 10 7 cells per two spleens.
Note: The frequency of CD4 + T cells in splenic white blood cells decreases with age. We found that the mean frequency G standard error of the mean (SEM) was 16.74% G 0.52% in mice at 43-50 weeks of age (n = 8 mice) in contrast to 23.78% G 0.89% at 9 weeks of age (n = 5 mice).
Note: This purification step of CD4 + T cells allows the isolation of CD153 + SA-T cells in a shorter time during a cell sorting step. CD4 MicroBeads do not interfere with anti-CD4 Ab (clone GK1.5) in the staining panels (Tables 1 and 2) and thus are compatible.  a. Straight gating strategy. i. Make the plots and gates as shown in Figure 2A.

Isolation and stimulation of CD153 + SA-T cells
ii. Load the sample tube containing the cells stained with panel A.
i. Make the plots and gates as shown in Figure 2B.
ii. Load the sample tube containing the cells stained with panel B.
Note: Set the sample temperature control at 4 C. A high temperature may cause the downregulation of Ab-labeled surface antigens.
Note: Sort a sufficient number of the cells (if possible, at least 1.5 times more cells than the actual number you need).
13. During or at the end of sorting, confirm the successful sorting. a. Unload the sample tube. b. Mix the collecting medium well with a sterile 1000 mL tip and add a small aliquot (e.g., 200 mL) to a 5 mL polystyrene tube.

OPEN ACCESS
c. Reload the aliquot and confirm that almost all of the cells are plotted within or close to the target gate ( Figure 2C). 14. Take a note of the ''total sort count'' displayed on the Sort Layout Window. 15. Bring the Ab-coated culture plate at 4 C to room temperature (20 C-25 C). 16. Centrifuge the sorted cells.
a. Centrifuge the sorted cells at 350 3 g for 15 min at 4 C. b. Aspirate the supernatant. c. Repeat a for 5 min and b to further remove the supernatant. d. Suspend the cells in culture medium to an estimated concentration of 2 3 10 6 cells/mL based on the ''total sort count'' (e.g., use 300 mL medium for 6 3 10 5 counts).
Note: After the first centrifuge and aspiration step (16a, b), a small but non-negligible volume of the supernatant will still remain on the surface of the tube. The second step (16c) allows almost complete removal of the supernatant and then makes the adjustment of the cell concentration accurate at step 18.
17. Count the actual cell number. a. Take 10 mL of cell suspension and mix it with 10 mL Trypan Blue Stain (0.4%) by pipetting. Inject 10 mL of the mixed cell suspension into a hemocytometer. b. According to the manual, count the unstained alive cells on a microscope and calculate the actual concentration of the cells.
Note: Almost all of the cells should be alive at this point. The actual concentration is usually somehow lower than the estimation due to a possible loss (e.g., attachment of the cells to the collection tube). See problem 2 and 3 in the troubleshooting section for more detail.
18. Adjust the concentration to 1 3 10 6 cells/mL by adding culture medium. 19. Initiate the stimulation with Abs. a. Dilute anti-CD28 Ab 1:250 in culture medium to make a 23 (4 mg/mL) anti-CD28 Ab solution. b. Using a 10 mL tip, aspirate the coating solution and PBS from the Ab-coated plate. c. Add 50 mL of 23 anti-CD28 Ab solution and culture medium to the corresponding wells (the former to the wells in blue and magenta colors and the latter to the wells in yellow color in Figures 1A and 1B). d. Add 50 mL of the cell suspension to the corresponding wells; now each well contains 100 mL.
Note: Aspiration should be performed from the edge of the well. There is no need to mix by pipetting after adding the cell suspension. 20. Incubate the plate at 37 C in a humidified 5% CO 2 incubator.
Note: As necessary, use the remaining cells for other assays such as cytokine FACS, in vitro survival assays, and RNA extraction.

Timing: 3 days for incubation and 4 h for assessment
The proliferation capacity is assessed by FACS.

After a 72-h incubation, harvest the cells and supernatant.
a. Suspend the cultured cells by gentle pipetting using a multichannel pipette and transfer them to a 96-well round bottom plate (the lid of the culture plate is reused in following steps). b. Centrifuge the plate at 400 3 g for 5 min. Note: It is highly recommended to observe the cells under a microscope before harvesting. T cell activation is manifested by enlargement in cell size and apparent cell growth.
Note: The supernatant should be stored in aliquots to avoid freeze-thaw cycles. Vortexing the plate should be performed carefully to prevent the cells from scattering away from the wells.
Alternatives: Although the proliferation capacity is assessed by the expression of Ki67, a marker of cell cycle progression, it is also useful to evaluate cell growth with a commercially available kit using an aliquot of the sample.  Figures 3A and 3B). See also problems 4 and 5 in the troubleshooting section. Note: Unlike FACS analysis of the cell surface antigens on viable cells, do not add PI.

Assessment of OPN production
Timing: 1 day ($24 h) OPN production is assessed by ELISA using a commercially available kit (R&D).

EXPECTED OUTCOMES
According to the isolation protocol here, we obtained about 7.5 3 10 5 cells of CD153 + SA-T cells from two spleens of mice at about 12 months of age. Note that because CD44 hi CD4 + T cells express gradually varying levels of PD-1 and CD153 (Figures 2A and 2B), the number available is affected by how strictly CD153 + PD-1 + cells is gated on a flow cytometry plot.
The FACS analyses of Ki67 ( Figures 3A and 3B) showed that 9.94% G 3.10% of CD153 + SA-T cells expressed Ki67 upon stimulation by anti-CD3ε/CD28 Abs in contrast to more than 70% of PD-1 -CD44 hi cells. 1 By anti-CD3ε/CD28/CD153 Abs, the frequency of Ki67 + in CD153 + SA-T cells was increased to 33.34% G 6.25%. 1 Besides Ki67 expression, T cell activation was manifested by an enlargement in cell size, which was also confirmed in the same FACS analyses (Figures 3A and 3B).

LIMITATIONS
This protocol isolates CD153 + SA-T cells from the spleens based on their constitutive expression of PD-1 and CD153. However, these surface antigens can be transiently induced in vitro upon stimulation, 9,10 suggesting that the activated T cells transiently expressing these antigens may also be present in vivo and such cells would be indistinguishable from authentic CD153 + SA-T cells in this protocol.
An intimate interaction between T cells and antigen-presenting cells allow the appropriate antigen recognition and a subsequent T cell response. A TCR complex consisting of variable TCRab and invariable CD3 chains (g, d, ε, and z) recognizes the antigen presented on a major histocompatibility complex and evokes signaling cascades to activate T cells. The other signaling cascades downstream of CD28 serves to ensure the TCR-mediated response. 11 Additionally, a number of molecules including CD153, immune checkpoint receptors, and even unrevealed ones may participate in the regulation of TCR signaling. Stimulation by plate-bound anti-CD3ε with soluble anti-CD28 Abs is very useful way to evoke TCR/CD28 signaling in vitro and is employed for investigating T cell function. However, it is worth noting that this protocol is simplified compared to the possible complexity of the molecular interactions in vivo and thus may not be fully identical. In a similar context, although plate-bound anti-CD153 Ab recapitulates the enhanced TCR-mediated activation of CD153 + SA-T cells, 1 whether it entirely recapitulates the activation mechanism needs to be considered.
Besides the stimulatory Abs, the mixed lymphocyte reaction (MLR) may allow evaluation of the response of CD153 + SA-T cells to antigen-presenting cells. CD153 + SA-T cells cultured in direct contact with B cells, both of which were isolated from lupus-prone mice, produced more OPN compared to those without B cells. 3

Problem 1
Frequency of CD153 + SA-T cells appears to be low or high compared with that in the literature.

Potential solution
Note that the frequency may be affected by mouse strains, housing conditions, and sex and that aged mice may occasionally exhibit considerable interindividual difference in the number of CD4 + T cell subpopulations. If necessary, assess the basal frequency in the experimental settings using a sufficient number of mice. If it is still too low, check the compensation of your data. For example, be careful of compensation between PE-labeled CD153 and PI because incorrect compensation may push the CD153 + cells away from a PIviable cell gate.

Problem 2
Dead cells are frequently found after cell sorting.

Potential solution
Check the gating strategy in which dead cells should be excluded on the basis of PI fluorescence.
Prepare new buffers and culture medium. During the purification and isolation steps, the cells should be kept at 4 C or on ice.

Problem 3
The yield of CD153 + SA-T cells after cell sorting is low.

Potential solution
Confirm that you have used a ''polypropylene'' 15 mL tube for collection, and coat the whole surface inside the tube with medium before sorting. Centrifuge the sorted cells for a longer time (e.g., 15 min) than usual. Nevertheless, the number of the sorted cells will probably decrease during the following procedures. A simple solution to increase the yield is to use more mice and pool their spleens.

Problem 4
Unexpectedly, CD153 + SA-T cells appear to proliferate substantially and secrete typical T cell cytokines rather than OPN in response to anti-CD3ε and anti-CD28 Abs.

Potential solution
Carefully confirm whether the compensation is adjusted correctly. Incorrect compensation may push the PD-1cells into a PD-1 + cell fraction. Considering the primary finding that CD153 + SA-T cells are increased with age, it is preferable to isolate the accumulated CD153 + SA-T cells from mice at or after middle age (R12 months) rather than collect a small number of cells from younger mice; this will reduce the likelihood of heterogeneity caused by transiently activated cells (see Limitations).

Problem 5
Additional presence of plate-coated anti-CD153 Ab does not enhance Ki67 expression and OPN production of enriched CD153 + SA-T cells upon TCR stimulation.

Potential solution
Ensure successful enrichment by staining an aliquot of the sorted cells with both PE-labeled anti-CD153 Ab and isotype-matched control IgG. Successful enrichment will manifest as about 75% purity of CD153 + cells. For plate coating, use anti-CD153 Ab clone RM153, since the epitope may also ll OPEN ACCESS be responsible for the successful enhancement. It is highly recommended that, according to the present protocol, anti-CD3ε or anti-CD153 Ab not be used at the isolation step of CD153 + SA-T cells, because these Abs may mask CD3ε or CD153 and consequently interfere with the plate-bound Abs at a later stimulation step.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Yuji Fukushima (fukushima.yuji.4x@kyoto-u.ac.jp).

Materials availability
This study did not generate new unique reagents.

Data and code availability
This study did not generate new datasets and code.